The metabolic sensor AMP-activated kinase (AMPK) detects the cellular energy status and adjusts metabolic activity according to the ATP:AMP ratio. AMPK is colocalized with ion channels such as CFTR and epithelial Na⁺ channels (ENaC) in the apical membrane of epithelial cells and affects the function of these proteins. We analyzed the precise mechanism of action and significance of AMPK-regulation. CFTR normally remains closed at baseline, but nevertheless opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (S737 and S768) in the R-domain, formerly identified as 'inhibitory' PKA sites. Replacement of both serines by alanines i) reduced phosphorylation of the R-domain, with S768 having dramatically greater impact, ii) produced CFTR channels that were partially open in the absence of any stimulation, iii) significantly augmented their activation by IBMX/forskolin and iv) eliminated CFTR inhibition post AMPK activation. Our data also imply that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in non-stimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity. Using AMPKa1-/- mice and wild type littermates, we demonstrate regulation of CFTR by AMPK in native airways and intestine. There was no effect on Ca2⁺ mediated Cl^- secretion, activated by ATP or carbachol. Moreover CFTR-dependent Cl^- secretion was enhanced in the colon of AMPKa1-/- mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl^- secretion mediated by CFTR is controlled by AMPK in vivo. Na+ absorption by ENaC is also a highly energy-consuming process that is inhibited by AMPK. We show that the catalytic subunit a1 inhibits ENaC in epithelial tissues from airways, kidney and colon and that AMPK- regulation of ENaC is absent in AMPKa1-/- mice. These mice demonstrate enhanced electrogenic Na+ absorption that leads to subtle changes in intestinal and renal function and may also affect Na⁺ absorption and mucociliary clearance in the airways. We demonstrate that AMPK uses the ubiquitin ligase Nedd4-2 to inhibit ENaC by increasing ubiquitination and endocytosis of ENaC. Thus, enhanced expression of epithelial Na+ channels was detected in colon, airways, and kidney of AMPKa1-/- mice. Therefore, AMPKa1 is a physiologically important regulator of electrogenic Na⁺ absorption by ENaC and Cl^- secretion by CFTR and may provide a novel pharmacological target for controlling epithelial ion transport by both ion channels.

Supported by DFG SFB699A6/A7